What causes iPSC death after Passaging?

05 June 2023 7 3K Report

I am working with DF-19 iPSC, I noticed that my cells attach just fine on vitronectin-coated plate on the first day. However, a day after, I noticed that my cells undergo massive death. I have tried different conditions, I still cannot figure out why.

I use this protocol below:

-I Aspirate medium and wash with DPBS

-Detach with accutase (1ml/well), incubate for 3 mins

-Rinse cells with 3mL medium and transfer to the conical tube

- Determine viable cell density, centrifuge , aspirate the supernatant and resuspend in fresh medium (E8+supplement).

- I add 1.5mL E8 medium + ROCK inhibitor (Y27632) 15uL per well of 6 well plate. I seed at 1X10^5

Is there something I am doing wrong ? The image attached is a day after seeding, however, It gets worse after.

Ayodeji Abijo

Thank you Ziadoon Al-Akashi

for your brilliant comment. My vitronectin coated plates worked just fine for another person, however, I will look into that.

1.5mL medium to 15 μL of ROCK inhibitor (10μM ) per well

Fatemeh Etezadi

When do you change the medium? every single day or other day?

Let me know the coating protocol. type of vitronectin, concentration, incubation time, and temperature

Ayodeji Abijo

Hi Fatemeh Etezadi medium change is daily

Coating protocol

Initial concentration of 250μg/mL

12.5μg/50uL final concentration of vitronectin/PBS to 4 wells of 6-well plate

Vitronectin XF™

incubation time is usually 1-3 mins, 37oC

Thanks.

Fatemeh Etezadi

Did you incubate for 1-3 minutes? or 1-3 hours?

From my point of view, if you incubated for 1-3 minutes, you did not let the vitronectin attach to the cell culture plate, you need at least 1 hour of incubation.

You need 10ug/ml for the final concentration, not 250 ug/ml.

you need at least 1-hour incubation at RT (Room Temperature).

Please follow these steps if the vitronectin XF is purchased from stem cell technology:

https://www.stemcell.com/how-to-coat-cultureware-for-pluripotent-stem-cell-culture.html

Thaw Vitronectin XF™ at room temperature (15 - 25°C).

Using a 50 mL polypropylene conical tube, dilute Vitronectin XF™ in CellAdhere™ Dilution Buffer to reach a final concentration of 10 µg/mL (i.e. use 40 µL of Vitronectin XF™ per mL of CellAdhere™ Dilution Buffer).

Gently mix the diluted Vitronectin XF™. Do not vortex.

Immediately use the diluted Vitronectin XF™ solution to coat non-tissue culture-treated cultureware.

Gently rock the cultureware back and forth to spread the solution evenly across the surface. Note: If the cultureware surface is not fully coated by the Vitronectin XF™ solution, it should not be used for human ES or iPS cell culture.

Incubate at room temperature (15 - 25°C) for at least 1 hour before use. Do not let the Vitronectin XF™ solution evaporate. Note: If not used immediately, the cultureware must be sealed to prevent evaporation of the Vitronectin XF™ solution (e.g. with Parafilm®) and can be stored at 2 - 8°C for up to 1 week after coating. Allow stored coated cultureware to come to room temperature for 30 minutes before proceeding to step 7.

Gently tilt the cultureware onto one side and allow the excess Vitronectin XF™ solution to collect at the edge. Remove the excess solution using a serological pipette or by aspiration. Ensure that the coated surface is not scratched.

Wash the cultureware once using CellAdhere™ Dilution Buffer (e.g. use 2 mL/well if using a 6-well plate).

Aspirate wash solution and add your desired cell culture medium (e.g. 2 mL/well if using a 6-well plate).

Fatemeh Etezadi

BTW, if you are not forced to use vitronectin for coating, I highly recommend this protein coating below for hiPSC culturing. It is easy to use and super user-friendly. I have been working with this product for a long time and recommend it:

https://www.amsbio.com/imatrix-recombinant-laminin-511/

Good luck!

Ayodeji Abijo

Thank you Fatemeh Etezadi I didnt get your question about coating at first. i usually coat and store few days before I use. I think its not a coating issue because another colleague used my vitronectin-coated plates and the cells attached well. I would check out the protocol you sent and try to optimize. Thank you very much

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